Experimental Evidence for Noncanonical Thymine Cation Radicals in

Article pubs.acs.org/JPCB

Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

Experimental Evidence for Noncanonical Thymine Cation Radicals in the Gas Phase Andy Dang,† Huong T. H. Nguyen,† Heather Ruiz,‡ Elettra Piacentino,‡ Victor Ryzhov,*,‡ and František Tureček*,† †

Department of Chemistry, University of Washington, Bagley Hall, Box 351700, Seattle, Washington 98195-1700, United States Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States

‡

S Supporting Information *

ABSTRACT: Thymine cation radicals were generated in the gas phase by collisioninduced intramolecular electron transfer in [Cu(2,2′:6,2″-terpyridine)(thymine)]2+• complexes and characterized by ion−molecule reactions, UV−vis photodissociation action spectroscopy, and ab initio and density functional theory calculations. The experimental results indicated the formation of a tautomer mixture consisting chiefly (77%) of noncanonical tautomers with a C-7-H2 group. The canonical 2,4-dioxo-N-1,N-3-H isomer was formed as a minor component at ca. 23%. Ab initio CCSD(T) calculations indicated that the canonical [thymine]+• ion was not the lowest-energy isomer. This contrasts with neutral thymine, for which the canonical isomer is the lowest-energy structure. Exothermic unimolecular isomerization by a methyl hydrogen migration in the canonical [thymine]+• ion required a low energy barrier, forming a C-7-H2,O-4-H isomer. Noncanonical thymine tautomers with a C-7-H2 group were also identified by calculations as low-energy isomers of 2′-deoxythymidine phosphate cation radicals. The relative energies of thymidine ion isomers were sensitive to the computational method used and were affected by solvation. The noncanonical [thymine]+• ions have extremely low adiabatic recombination energies (REadiab < 5.9 eV), making them potential ionization hole traps in ionized nucleic acids.

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INTRODUCTION DNA ionization triggers degradation chemical reactions that can lead to DNA damage such as nucleobase loss and strand breaks.1 The processes involved in DNA ionization have been studied at several levels, ranging from individual nucleobases through nucleotides to double-stranded oligonucleotide DNA models.2−4 A phenomenon of particular interest has been the propagation of the cation-radical defect (a “hole”) from the initial site, generated randomly at a nucleobase, along the DNA strand. Model studies have tremendously contributed to our understanding of the hole migration at the nanoscale level. Nucleobase cation radicals have been generated by targeted photooxidation by intercalated transition metal complexes,5 organic groups,6 or photoinduced electron transfer.7 Among the DNA nucleobases, thymine has the highest ionization energy (8.8−9.4 eV), as established by photoelectron spectroscopy,8−10 photoionization,11,12 and computational studies.13−16 Perhaps even more relevant for solution studies, thymine also has the highest electrochemical oxidation potential among the DNA nucleobases.17,18 These properties make the thymine cation radical a likely electron acceptor by transfer from other nucleobases, and because of its high ionization potential, thymine might be considered a nonreactive spacer in studies of hole propagation by hopping between guanine bases.6 In contrast, recent studies of A-T-rich DNA models19−24 demonstrated the high reactivity of the ionized thymine nucleobase. Reaction of thymidine with photoexcited 2-methyl-1,4-naphthoquinone25,26 in the presence of molecular © XXXX American Chemical Society

oxygen gave intermediates that were hydroperoxylated at the C-5 methyl group, indicating deprotonation followed by oxygen addition to the C-7-H2 radical.27 The methyl group is also a preferred site of attack by OH radicals, forming thymine C-7-H2 radicals.28 The reactivity of thymine cation radicals has been addressed by density functional theory (DFT) calculations in studies that focused on intra- and intermolecular proton transfer.29−31 An important finding in these studies was the theoretical existence of a low-energy tautomer of [thymine]+• that was separated from the canonical 2,4-dioxo form by a relatively low-energy barrier.29 Thymine cation radicals have been produced as transient species by photoionization32,33 and radiolytic34−36 or chemical oxidation37−40 in solution, where they were exposed to solvents, additives, and side-reaction products. In contrast, the rarefied gas phase provides an ideal medium for studying isolated reactive species to investigate their intrinsic properties in the absence of solvent, counterions, and surfaces.41 Among nucleobase radicals, hydrogen adducts of cytosine, adenine, and uracil have been generated in the gas phase by femtosecond electron transfer to the corresponding cations, and their structure, energetics, and unimolecular dissociations have been investigated.42−44 Synthetic routes to gas-phase biomolecular cation radicals also Received: October 5, 2017 Revised: November 19, 2017 Published: December 8, 2017 A

DOI: 10.1021/acs.jpcb.7b09872 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B rely on reductive or oxidative electron transfer.45,46 For example, intramolecular oxidation of the biomolecule (M) in a complex with a transition metal (Cu2+, Fe3+) and an auxiliary ligand such as 2:2′,6′:2″-terpyridine (terpy) has been used to produce gas-phase peptide and nucleobase cation radicals.47,48 The oxidation is driven by collision-induced dissociation (CID) of a doubly charged complex generated by electrospray ionization and selected by mass, according to the equation (1)

gas-phase ion that are used for structure assignment. In particular, action spectroscopy has been used to elucidate structures of isomeric cytosine cation radicals50 and deoxyadenosine dinucleotides.46 Here, we report the generation and structure elucidation of isomeric thymine cation radicals. The high ionization energy of thymine represents a potential challenge for the efficient generation of thymine cation radicals.54 We show that thymine cation radicals produced in the gas phase exist in unexpected, low-energy tautomeric forms of potential relevance for the process of DNA ionization.

Gas-phase chemistry analogous to eq 1 has been used to generate cation radicals of 9-methylguanine49 and cytosine,50 as well as the more complex systems of deoxyguanosine51 and the Watson−Crick cytosine−guanine pair.52 The mass-selected cation radicals generated by eq 1 chemistry have been characterized by specific radical and electron transfer ion− molecule reactions, 50 infrared multiphoton dissociation (IRMPD) action spectroscopy,49−52 and single-photon UV−vis photodissociation action (UVPD) spectroscopy.50 The action spectroscopy methods rely on resonant photon absorption by a gas-phase ion to induce dissociation (“action”), whereby the fragment ions formed are detected by mass spectrometry.53 Action spectra can provide a detailed characterization of vibrational modes (IRMPD) or electronic excitations (UVPD) of the

EXPERIMENTAL SECTION Materials. Thymine, thymine-6,7,7,7-d4, 2′-deoxythymidine, Cu(NO3)2, and 2,2′:6′,2″-terpyridine were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Stock solutions (1 mg mL−1) of thymine and terpy were prepared in methanol, and those of thymidine and Cu(NO3)2 were made in water/methanol (50:50). The stock solutions of terpy and Cu(NO3)2 were combined in a 1:1 ratio and diluted 10-fold in methanol. An aliquot of this solution was combined with the thymine or thymidine solution, vortexed, and allowed to react for 10 min at room temperature. The resulting solution was diluted with methanol and used for direct infusion into the electrospray ion source.

[Cu(terpy)(M)]2 +• → [Cu(terpy)]+ + M+•

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Figure 1. Collision-induced dissociation MS2 spectra of (a) [Cu(terpy)(thymine]2+• at m/z 211 and 212, (b) [Cu(terpy)(thymine-1,3-d2)]2+• at m/z 212 and 213, and (c) [Cu(terpy)(thymine-6,7,7,7-d4)]2+• at m/z 213 and 214. B


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The Journal of Physical Chemistry B Scheme 1. Formation of [Thymine]+• Ions from Cu-terpy Complexes

Methods. Mass spectra were measured on an LTQ-XL-ETD linear ion trap (Thermo Electron Fisher, San Jose, CA) furnished with a home-built microspray electrospray ion source. Photodissociation action spectra were measured as described previously.55 Briefly, a laser beam from an EKSPLA NL301G Nd:YAG laser source (Altos Photonics, Bozeman, MT) was processed and mixed by an optical parametric oscillator (PG142C, Altos Photonics), and the output was focused into the linear ion trap to drive photodissociation of stored mass-selected [thymine]+• ions. The laser optical parametric oscillator (OPO) operates at 20 Hz with a 3−6-ns pulse width. The laser power (0.52−12.69 mJ per pulse peak) was measured at each wavelength and used to normalize the photofragment ion intensities. The spectra were reproduced twice on different days. High-resolution mass spectra were measured on an Orbitrap Velos mass spectrometer (Thermo Electron Fisher) at a resolving power of 60000. Ion−molecule reactions of massselected [thymine]+• ions were measured on a modified Bruker Esquire 3000 quadrupole ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) as described previously.56 Furan, anisole, water, and acetic acid were introduced through a leak valve. The reaction time was defined by the delay between the ion isolation and product scans. Calculations. Standard ab initio and density functional theory calculations were performed with the Gaussian 09 suite of programs.57 The thymine ion and neutral geometries were optimized with the B3LYP,58 M06-2X,59 and ωB97X-D60 hybrid DFT methods using the 6-311+G(2d,p) basis set. Local energy minima and first-order saddle points were characterized by harmonic frequency analysis as having the appropriate numbers of imaginary frequencies. Solvation energies were calculated with the M06-2X and ωB97X-D methods using the polarizable continuum model61 available in Gaussian 09. Single-point energies were calculated using Møller−Plesset perturbation theory62 truncated at second order [MP2(frozen core)] with the aug-cc-pVTZ basis set.63 The MP2 energies were corrected for spin contamination by annihilation of higher spin states to provide spin-projected (PMP2) energies.64,65 Alternatively, restricted open-shell calculations (ROMP2) were used for radicals.66 Another set of single-point energies was obtained by coupled-cluster calculations with single, double, and disconnected

Figure 2. Kinetics of charge exchange between [thymine]+• (m/z 126) and anisole, forming [anisole]+• at m/z 108.

Figure 3. UV−vis photodissociation action spectrum of [thymine]+• from [Cu(terpy)2′-thymine]2+•.

triple excitations [CCSD(T)]67,68 using the aug-cc-pVDZ and 6-311++G(3df,2p) basis sets. The CCSD(T)/aug-cc-pVDZ calculations were expanded to aug-cc-PVTZ using the linear relationship E[CCSD(T)/aug-cc-pVTZ] ≈ E[CCSD(T)/aug-ccpVDZ] + E[PMP2/aug-cc-pVTZ] − E[PMP2/aug-cc-pVDZ]. C


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The Journal of Physical Chemistry B

of [Cu(terpy)thymine]2+• ions that were formed by the electrospray of water/methanol solutions containing equimolar concentrations of Cu(NO3)2, terpy, and thymine (Figure 1a). The Cu complex was isolated as a mixture of 63Cu and 65Cu isotopologues, and its CID resulted in a major loss of neutral thymine, forming the [Cu(terpy)]2+• ions (m/z 148 and 149 for the 63Cu and 65Cu isotopes, respectively; 64%) and the corresponding water adducts at m/z 157 and 158 (30%). The competitive collision-induced intramolecular electron transfer in [Cu(terpy)thymine]2+• yielded the complementary [thymine]+• (m/z 126) and [Cu(terpy)]+ (m/z 296, 298) ions that were produced in a 6% relative fragment yield. The identity of the [thymine]+• ion was supported by accurate mass measurements (measured 126.0427, theoretical 126.0424 for C5H6N2O2+•, error < 2.5 ppm). The branching ratio of the competing channels changed only very slightly (±0.2%) with the applied collision energy. CID with electron transfer provided a sufficient [thymine]+• ion count for further multistep tandem mass spectrometry (MSn) studies. H/D exchange in D2O/CD3OD solution and electrospray were used to generate [Cu(terpy)(thymine-d2)]2+• precursor ions, and upon mass isolation and CID, [thymine-d2]+• ions were obtained (Scheme 1, Figure 1b). [Thymine-d4]+• ions were prepared analogously in 5.7% yield from mass-selected [63Cu(terpy)(thymine-d4)]2+• and [65Cu(terpy)(thymine-d4)]2+• complexes using thymine-6,7,7,7-d4 as the ligand (Figure 1c). The [thymine]+• ions and their d2 and d4 analogues were characterized by multistep tandem mass spectrometry (CID-MS3) spectra that showed loss of HNCO and (HNCO + CO) from [thymine]+• and [thymine-6,7,7,7-d4]+• (Figure S2a−c, Supporting Information). These are also major dissociations upon electron ionization of gaseous thymine.78−81 CID-MS3 of [thymine-1,3-d2]+• resulted in loss of DNCO and HNCO in a 86:14 ratio (Figure S2b). Ion−Molecule Reactions. The mass-selected and thermalized (300 K) [thymine]+• ions were probed by ion−molecule

Excited states were treated by equation-of-motion- (EOM-) CCSD calculations69,70 using the 6-31+G(d,p) basis set. Further sets of excitation energies and oscillator strength values were obtained by time-dependent DFT (TD-DFT) calculations71 with the M06-2X and ωB97X-D methods using the 6-31+G(d,p) and 6-311+G(2d,p) basis sets. TD-DFT calculations of polyatomic radicals have been found to show only a very weak dependence on the basis set.50,72,73 This was also the case for [thymine]+•, where excitation energies calculated by TD-DFT for 20 excited states with the 6-31+G(d,p) and 6-311+G(2d,p) basis sets showed a close correlation (r 2 = 0.9997) and small root-mean-square deviations [rmsd(ΔEexc) = 0.046 eV, rmsd(λ) = 3.8 nm]. In contrast, EOM-CCSD-calculated excitation energies depend on the basis set, exhibiting a 0.1−0.2 eV downward shift upon use of a larger basis set compared [6-311++G(2d,p)] to 6-31+G(d,p).72 Comparison of the TD-DFT and EOM-CCSD excitation wavelengths indicated a close correlation of the main lines (Figure S1, Supporting Information). The M06-2X excitations showed a red shift that was coincidentally of similar magnitude as that observed using EOM-CCSD calculations with a larger basis set. Therefore, TD-DFT M06-2X/6-31+G(d,p) calculations were used to calculate electronic transitions in multiple Boltzmann-weighed configurations of thermal (300 K) ions using the Newton-X program.74 This generated vibronically corrected absorption spectra that are discussed later in this article. Rice−Ramsperger−Kassel− Marcus (RRKM) calculations of unimolecular rate constants75 were performed using the QCEP program76 that was recompiled for MS-DOS77 and run under Windows 7. Rotational states were treated adiabatically, and the microcanonical rate constants k(E,J), were Boltzmann averaged over the rotational state distribution at >298 K.

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RESULTS AND DISCUSSION Generation of Thymine Cation Radicals. Thymine cation radicals were produced by collision-induced dissociation (CID) Table 1. Energies of Thymine Cation Radicals

relative energya (kJ mol−1) isomer

B3LYP/6-311+G(2d,p)

ωB97X-D

M06-2X

CCSD(T)/6-311++G(3df,2p)b

CCSD(T)/aug-cc-pVTZb,c

1 2+• 3+• 4+• 5+• 6+• 7+• 8+• 9+• 10+• 11+• 12+• 13+• TS1 TS2 TS3 TS4 TS5 TS6 C4H5NO+• + OCNH C3H5N+• + CO + OCNH C3H5N+• + OCNCHO

d

d

d

d

0 (0)d 12 42 40 44 57 80 −34 (−29) −12 (−8) −7 (−4.1) 2 (6) 155 185 107 136 131 187 175 200 193 208

+•

0 (0) 13 32 45 49 58 64 −26 (−21)d −10 (−7) 1.5 (4.8) 10 (15) 158 174 104 143 140 170 172 264 187 182 182

0 (0) 13 38 47 50 60 67 −25 (−21) −9 (−6) 1.9 (5.2) 13 (17) 181 192 110 146 144 207 190 263 208 228 222

0 (0) 6 30 33 36 55 58 −41 (−36) −15 (−12) −6 (−3) −3 (1.3) 179 189 111 137 134 207 189 250 216 212 217

0 (0) 15 45 46 −28 (−24) −8 (−5) −5 (−1.6) 8 (12) 157 189 113 144 139 191 l80 198 192 207

a Including B3LYP/6-311+G(2d,p) zero-point energy corrections and referring to 0 K unless stated otherwise. bSingle-point energies on MP2(full)/ 6-31G(d,p)-optimized geometries. cFrom basis set expansion of aug-cc-pVDZ → aug-cc-pVTZ. dValues in parentheses are relative free energies at 310 K.

D


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m/z 83 and m/z 55 fragment ions, respectively. Note that these fragments were also observed upon CID of [thymine]+• (Figure S2a). The wavelength profiles of the relative intensities of the m/z 83 and m/z 55 fragment ions overlapped but showed different maxima. The m/z 83 ion exhibited a broad band with a maximum at 520 nm. The m/z 55 ion exhibited multiple bands: a weak broad band with a maximum at 580, bands at 460 and 350 nm, and a strong UV band at 260 nm. The bands in the visible and near-UV regions are absent in the UV spectrum of neutral and protonated thymine83 and represent a signature of the cation-radical chromophores. To interpret the action spectrum of [thymine]+•, we performed extensive ab initio calculations of isomeric ion structures and excitation energies, as described in the next sections. Thymine Cation Radical Structures. Thymine cation radicals can form several tautomers including those in which a hydrogen from the CH3 group has migrated into a ring position.29 We obtained optimized structures and DFT relative energies of 18 [thymine]+• isomers (Table 1), and 11 low-energy structures were evaluated by CCSD(T) single-point energy calculations. Structures of the low-energy isomers are shown in Figure 4; those of the others (14+•−20+•) are shown in Figure S5

reactions. Exothermic charge-transfer ion−molecule reactions typically proceed at the collision rate82 and provide a sensitive probe of ion structure in situations where isomeric ions differ in their adiabatic recombination energies (REs).50 Previous photoelectron spectroscopic measurements8−10 established the adiabatic ionization energy of gas-phase thymine as 8.8−9.40 eV, which is equal to the absolute value of the RE. The gas-phase charge-exchange reaction of [thymine]+• with gaseous furan (IE = 8.88 eV) did not proceed at a measurable rate, as evidenced by the fact that 8.42 eV, whereas the major fraction of isomers had low recombination energies preventing charge transfer to anisole. To investigate these new isomers, we performed additional ion−molecule reactions investigating proton transfer of [thymine1,3-d2]+• and [thymine-6,7,7,7-d 4]+• with gaseous water and acetic acid. [Thymine-1,3-d2]+•, with a canonical 2,4-dioxo structure, has the N-1-D and N-3-D in exchangeable positions and was expected to undergo gas-phase D/H exchange, gradually forming [thymined1]+• and [thymine]+• ions. Figure S3 shows the time course of the D/H exchange reaction with residual water that was measured in the linear ion trap and can be expressed by the following pseudofirst-order kinetic equation: I(thymine-d2) = I0e−0.135t with rmsd = 2.3%. These experiments established that the water partial pressure in the ion trap was sufficient to promote D/H exchange in exchangeable positions of the trapped ions. In contrast, the canonical 2,4-dioxo isomer of [thymine6,7,7,7-d4]+• has the deuterium in nonexchangeable C−D positions and was not expected to undergo D/H exchange with water vapor. Nevertheless, the [thymine-6,7,7,7-d4]+• ion was found to undergo slow D/H exchange in the gas-phase reaction with water and acetic acid, forming the d3 isotopologue (Figure S4). This result indicates that a fraction of the [thymine-6,7,7,7-d4]+• ions formed from the [Cu(terpy)]2+• complex did not have the canonical structure but rather underwent a prototropic rearrangement that moved the CD3 or 6-D atoms into an exchangeable position (O-2 or O-4). In addition, these isomers must have recombination energies of 260 kJ mol−1 less stable than 1 (Table 2). Neutral isomer 9 was found to be a transition state (TS) that collapsed to 1 upon O−H rotation. The high energies of neutral species 8, 10, and 11 combined with the low energies of the respective cation radicals resulted in extremely low (190 kJ mol−1, where the rate constants reached 14 s−1 for 50% reaction at 50 ms (Figure S7). The high threshold energy for the

ring opening explains why 1+• can undergo extensive isomerization by reversible hydrogen migrations to generate a mixture of nondissociating [thymine]+• ions. The RRKM kinetics also suggest that the dissociation of thermal [thymine]+• having a mean internal energy of 24 kJ mol−1 at 310 K can be promoted by an absorption of >176 kJ mol−1 energy, corresponding to a single 680-nm photon. Formation of [Thymine] +• from the [Cu(terpy)(thymine)]2+• Complex. The formation of unusual [thymine]+• isomers upon CID of the [Cu(terpy)(thymine)]2+• complex also raised the question of whether thymine coordination to Cu2+ in solution could lead to isomerization. We investigated by DFT calculations the relative energies of several [Cu(terpy)(thymine)]2+• complexes in which the thymine ligands were different tautomers of the neutral molecule (Figure 8, Table 3). According to these calculations, the complex with an O-4-linked 1, [Cu(terpy)(O-4-1)]2+•, was the lowestenergy structure in both the gas phase and aqueous and methanol solutions, followed by the complexes with an O-2-linked 1 and an O-4-linked 2. In contrast, Cu(terpy) complexes with 8, 9, and 10 were >120 kJ mol−1 less stable than [Cu(terpy)(O-4-1)]2+• and were unlikely to be present under equilibrium conditions in solution or formed by electrospray in the gas phase. We conclude that the formation of gas-phase 8+•−11+• most likely occurs as a result of collisional excitation and dissociation of the Table 3. Relative Energies of [Cu(terpy)(thymine)]2+• Complexes relative energya (kJ mol−1) complex

B3LYPb

M06-2Xb

ωB97X-Db

[Cu(terpy)→O-4-1]2+• [Cu(terpy)→O-2-1]2+• [Cu(terpy)→O-4-2]2+• [Cu(terpy)→N-1-2]2+• [Cu(terpy)→N-3-9]2+• [Cu(terpy)→C-7-9]2+• [Cu(terpy)→C-7-8]2+•

0 17 36 86 120 130 154

0 (0)c 16 (7) 7 (31) 76 (49) 145 (182) 128 (159) 159 (158)

0 (0) 20 (12) 19 (43) 78 (49) 125 (159) 125 (155) 145 (142)

a

Including B3LYP/6-311+G(2d,p) zero-point energy corrections and referring to 0 K. bCalculations with the 6-311+G(2d,p) basis set. c Values in parentheses include solvation energies in the polarizable continuum of water. H


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The Journal of Physical Chemistry B [Cu(terpy)(thymine]2+• complex that produces a fraction of [thymine]+• ions with internal energies of >128 kJ mol−1 for kinetically relevant isomerization. Thymidine Cation Radicals. We attempted to generate 2′-deoxythymidine cation radicals by collision-induced intramolecular electron transfer in the [Cu(terpy)(thymidine)]2+• complex. The precursor ion was successfully made by electrospray ionization (m/z 269 and 271 for the 63Cu and 65Cu isotopes, respectively). However, CID of [Cu(terpy)(thymidine)]2+• resulted in a dissociation of the N-glycosidic bond forming the complementary deoxyribosyl (C5H9O3+, m/z 117) and [Cu(terpy)(thymine)]+• (m/z 421) singly charged fragment ions (Figure S9a, Supporting Information). The major CID product was the [Cu(terpy)(thymine)]2+• ion (m/z 211), which, upon mass isolation and CID-MS3 gave the [thymine]+• ion, which, according to its UVPD and CID spectra was identical to the ion formed directly from the doubly charged thymine

complex (Figure S9b, Supporting Information). The unequivocal course of dissociation of the [Cu(terpy)(thymidine)]2+• complex indicates that the cleavage of the 2′-deoxythymidine glycosidic bond is energetically more favorable than the electron-transfer reaction forming [thymidine]+• and [Cu(terpy)]+ ions. Thymidine Isomerization in Ionized Nucleotides. The existence of low-energy [thymine]+• isomers could have a serious effect on charge transfer in ionized oligonucleotides. Because the recombination energies of 8+•−11+• are extremely low, in fact, much lower than the REadiab values of the other nucleobases, thymine ionization and isomerization would trap the hole at the thymine residue, providing a C-7-H2 radical group for thyminespecific reactions.19−24 To address this point, we investigated by DFT and ab initio computations the structures and relative energies of 2′-deoxythymidine phosphate cation radicals, both in the gas phase and in the polarizable dielectric of the surrounding water continuum. The ion relative energies, represented by the lowest-energy conformers 21+•−25+• (Figure 9), were found to depend on the computational method and also to be affected by entropies and solvent effects (Table 4). The canonical tautomer 21+• marginally had the lowest free energy (ΔG°g,298) among the 2′-deoxythymidine phosphate isomers in the gas phase, chiefly because of its high vibrational entropy at 298 K. However, the ΔG°g,298 values for 21+• and the C-7-H2 tautomers 23+• and 24+• were as close as 2 kJ mol−1 (Table 4). Solvation by water favored the C-7-H2 tautomer 22+•, whose ΔGg,298 ° value was very close to those of 21+• and 24+•. The calculations indicated that the relative stabilities of thymidine tautomers are extremely sensitive to the local environment and solvent. To resolve this issue, a dedicated computational study of cation-radical A−T pairs in oligonucleotides is needed; however, this exceeds the scope of this work. A conspicuous feature of thymidine cation-radical structures displaying strong intramolecular hydrogen bonds is the spontaneous migration of a thymine hydroxyl proton to the phosphate group, forming distonic ions 22+•−24+•. These ions are expected to behave as thymine radicals and to undergo typical reactions triggered by oxygen addition to the C-7-H2 group. The internal prototropic isomerization in 22+•−24+• is an alternative to external proton transfer from ionized thymine to the surrounding water molecules, as suggested previously.24

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CONCLUSIONS The experimental and computational results presented in this article point to the existence of noncanonical [thymine]+• isomers having the C-7-H2 group. The new isomers are formed as major products by intramolecular electron transfer in gasphase metal−thymine complexes and represent the lowestenergy structures that are more stable than the canonical 2,4dioxo-N-1,N-3-H form. Theoretical calculations also predict the

Figure 9. M06-2X/6-311+G(2d,p)-optimized structures of [2′-deoxythymidine phosphate]+• isomers.

Table 4. Relative Energies of [2′-Deoxythymidine phosphate]2+• Isomers relative energya (kJ mol−1) gas phase

solvated

b

B3LYP

M06-2X

ωB97X-D

B3LYP

M06-2X

ωB97X-D

21+• 22+• 23+• 24+• 25+•

0 (0)c 16 (28) 7 (19) 12 (22) 17 (25)

0 (0)c 3 (14) −4 (8) −8 (2) 10 (18)

0 (0)c 8 (20) 9 (21) 7 (16) 19 (27)

0 (0)c 3 (14) 23 (33) 27 (34) 28 (29)

0 (0)c −6 (5) 13 (22) −1 (6) 23 (31)

0 (0)c −2 (9) 25 (35) 19 (26) 31 (39)

ion

a

Including B3LYP/6-31+G(d,p) zero-point energy corrections and referring to 0 K unless stated otherwise. bLowest-energy conformers. cRelative free energies at 298 K. I


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existence of low-energy noncanonical nucleobase isomers of 2′-deoxythymidine phosphate cation radicals. It remains to be explored if such unusual species play a role in DNA ionization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b09872. Complete ref 57, M06-2X/6-311+G(2d,p)-optimized geometries and total energies of thymine and thymidine cation radicals, UV−vis absorption spectra, CID mass spectra, D/H exchange kinetic plots, and RRKM rate constants (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 815-753-6955. *E-mail: [email protected]. Phone: 206-685-2041. Fax: 206-685-8665. ORCID

František Tureček: 0000-0001-7321-7858 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Research at University of Washington was supported by the National Science Foundation Division of Chemistry (Grants CHE-1359810, CHE-1661815, and CHE-1624430) and the Klaus and Mary Ann Saegebarth Endowment. H.R. thanks the NSF-REU program (Grant CHE-1659548 to Northern Illinois University) for support.

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REFERENCES

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Experimental Evidence for Noncanonical Thymine Cation Radicals in

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